The determination of the catechol generating substance salicylate in biological fluids such as human serum, has diagnostic significance. Acetylsalicylic acid (aspirin) is used as an analgesic and as an anti-inflammatory drug for arthritis. It rapidly hydrolyzes to salicylate which has the therapeutic effect. The therapeutic level as an analgesic is up to 20 mg/dl. For arthritis the level is up to 30 mg/dl. Problems such as headaches, tinnitus, flushing and hyperventilation occur at higher salicylate levels followed by imbalances in the acid-base level. Salicylate levels above 60 mg/dl can be lethal.
One method for assaying salicylate employs the enzymatic conversion of salicylate to catechol catalyzed by salicylate hydroxylase with the accompanying conversion of NADH to NAD.sup.+. There is a quantitative correlation between disappearance of NADH, as reflected in a change in optical density at 340 nm, and the concentration of salicylate (You and Bittikofer, Clin. Chem., 30:1549, 1984). The problem is that this method is unsuitable for incorporation into a dry format to be stored over time because of the instability of NADH at low pH and the low extinction coefficient of NADH.
Other problems of existing methods for the determination of salicylate suffer from poor sensitivity of the NADH method, interferences from phenolic and ketoacids normally present in serum, Ferric chloride, time-consuming procedures, inability to measure protein bound salicylate (measure only free salicylate refers to ion selective electrodes), or unsuitability for dry format.